Sustained glucagon receptor antagonism in insulin-deficient high-fat-fed mice.

Discerning modification to the amino acid sequence of native glucagon can generate specific glucagon receptor (GCGR) antagonists, that include desHis1Pro4Glu9-glucagon and the acylated form desHis1Pro4Glu9(Lys12PAL)-glucagon. In the current study, we have evaluated the metabolic benefits of once-daily injection of these peptide-based GCGR antagonists for 18 days in insulin-resistant high-fat-fed (HFF) mice with streptozotocin (STZ)-induced insulin deficiency, namely HFF-STZ mice. Administration of desHis1Pro4Glu9-glucagon moderately (P < 0.05) decreased STZ-induced elevations of food intake. Body weight was not different between groups of HFF-STZ mice and both treatment interventions delayed (P < 0.05) the onset of hyperglycaemia. The treatments reduced (P < 0.05-P < 0.001) circulating and pancreatic glucagon, whilst desHis1Pro4Glu9(Lys12PAL)-glucagon also substantially increased (P < 0.001) pancreatic insulin stores. Oral glucose tolerance was appreciably improved (P < 0.05) by both antagonists, despite the lack of augmentation of glucose-stimulated insulin release. Interestingly, positive effects on i.p. glucose tolerance were less obvious suggesting important beneficial effects on gut function. Metabolic benefits were accompanied by decreased (P < 0.05-P < 0.01) locomotor activity and increases (P < 0.001) in energy expenditure and respiratory exchange ratio in both treatment groups. In addition, desHis1Pro4Glu9-glucagon increased (P < 0.01-P < 0.001) O2 consumption and CO2 production. Together, these data provide further evidence that peptidic GCGR antagonists are effective treatment options for obesity-driven forms of diabetes, even when accompanied by insulin deficiency.

Metabolic effects of combined glucagon receptor antagonism and glucagon-like peptide-1 receptor agonism in high fat fed mice.

Ablation of glucagon receptor (GCGR) signalling is a potential treatment option for diabetes, whilst glucagon-like peptide-1 (GLP-1) receptor agonists are clinically approved for both obesity and diabetes. There is a suggestion that GCGR blockade enhances GLP-1 secretion and action, whilst GLP-1 receptor activation is known to inhibit glucagon release, implying potential for positive interactions between both therapeutic avenues. The present study has examined the ability of sustained GCGR antagonism, using desHis1Pro4Glu9-glucagon, to augment the established benefits of the GLP-1 mimetic, exendin-4, in high fat fed (HFF) mice. Twice-daily injection of desHis1Pro4Glu9-glucagon, exendin-4 or a combination of both peptides to groups of HFF mice for 10 days had no impact on body weight or energy intake. Circulating blood glucose and glucagon concentrations were significantly (P < 0.05-0.01) decreased by all treatment regimens, with plasma insulin levels elevated (P < 0.001) when compared to lean control mice. Intraperitoneal and oral glucose tolerance were improved (P < 0.05-0.01) by all treatments, despite lack of enhanced glucose-stimulated insulin secretion. Following exogenous glucagon administration, all HFF treatment groups displayed reduced (P < 0.05-0.001) glucose and insulin levels compared to HFF saline controls, although peripheral insulin sensitivity was largely unchanged across all animals. Interestingly, all treatments had tendency to increase pancreatic insulin content with pancreatic glucagon content significantly elevated (P < 0.05) by all interventions. These studies highlight the capacity of peptide-based GCGR inhibition, or GLP-1 receptor activation, to significantly improve metabolism in HFF mice but suggest no obvious additive benefits of combined therapy.

Modelling pancreatic ß-cell inflammation in zebrafish identifies the natural product wedelolactone for human islet protection.

Islet inflammation and cytokine production are implicated in pancreatic ß-cell dysfunction and diabetes pathogenesis. However, we lack therapeutics to protect the insulin-producing ß-cells from inflammatory damage. Closing this clinical gap requires the establishment of new disease models of islet inflammation to facilitate screening efforts aimed at identifying new protective agents. Here, we have developed a genetic model of Interleukin-1ß (Il-1ß)-driven islet inflammation in zebrafish, a vertebrate that allows for non-invasive imaging of ß-cells and in vivo drug discovery. Live imaging of immune cells and ß-cells in our model revealed dynamic migration, increased visitation and prolonged macrophage retention in the islet, together with robust activation of NF-κB signalling in ß-cells. We find that Il-1ß-mediated inflammation does not cause ß-cell destruction but, rather, it impairs ß-cell function and identity. In vivo, ß-cells exhibit impaired glucose-stimulated calcium influx and reduced expression of genes involved in function and maturity. These defects are accompanied by α-cell expansion, glucose intolerance and hyperglycemia following a glucose challenge. Notably, we show that a medicinal plant derivative (wedelolactone) is capable of reducing the immune-cell infiltration while also ameliorating the hyperglycemic phenotype of our model. Importantly, these anti-diabetic properties in zebrafish are predictive of wedelolactone's efficacy in protecting rodent and human islets from cytokine-induced apoptosis. In summary, this new zebrafish model of diabetes opens a window to study the interactions between immune and ß-cells in vivo, while also allowing the identification of therapeutic agents for protecting ß-cells from inflammation.

Islet neuropeptide Y receptors are functionally conserved and novel targets for the preservation of beta-cell mass.

AIMS: Two unmet therapeutic strategies for diabetes treatment are prevention of beta-cell death and stimulation of beta-cell replication. Our aim was to characterize the role of neuropeptide Y receptors in the control of beta-cell mass. MATERIALS AND METHODS: We used endogenous and selective agonists of the NPY receptor system to explore its role in the prevention of beta-cell apoptosis and proliferation in islets isolated from both mouse and human donors. We further explored the intra-cellular signalling cascades involved, using chemical inhibitors of key signalling pathways. As proof of principle we designed a long-acting analogue of [Leu31 Pro34 ]-NPY, an agonist of the islet-expressed Y receptors, to determine if targeting this system could preserve beta-cell mass in vivo. RESULTS: Our data reveal that NPY Y1, 4 and 5 receptor activation engages a generalized and powerful anti-apoptotic pathway that protects mouse and human islets from damage. These anti-apoptotic effects were dependent on stimulating a Gαi-PLC-PKC signalling cascade, which prevented cytokine-induced NFkB signalling. NPY receptor activation functionally protected islets by restoring glucose responsiveness following chemically induced injury in both species. NPY receptor activation attenuated beta-cell apoptosis, preserved functional beta-cell mass and attenuated the hyperglycaemic phenotype in a low-dose streptozotocin model of diabetes. CONCLUSION: Taken together, our observations identify the islet Y receptors as promising targets for the preservation of beta-cell mass. As such, targeting these receptors could help to maintain beta-cell mass in both type 1 and type 2 diabetes, and may also be useful for improving islet transplantation outcomes.

Inhibitory effect of somatostatin on insulin secretion is not mediated via the CNS.

The inhibitory effect of somatostatin (SST) on insulin secretion in vivo is attributed to a direct effect on pancreatic beta cells, but this is inconsistent with some in vitro results in which exogenous SST is ineffective in inhibiting secretion from isolated islets. We therefore investigated whether insulin secretion from the pancreatic islets may partly be regulated by an indirect effect of SST mediated via the CNS. Islet hormone secretion was assessed in vitro by perifusion and static incubations of isolated islets and in vivo by i.v. or i.c.v. administration of the SST analogue BIM23014C with an i.v. glucose challenge to conscious, chronically catheterised rats. Hormone content of samples was assessed by ELISA or RIA and blood glucose levels using a glucose meter. Exogenous SST14/SST28 or BIM23014C did not inhibit the release of insulin from isolated rodent islets in vitro, whereas peripheral i.v. administration of BIM23014C (7.5 µg) with glucose (1 g/kg) led to decreased plasma insulin content (2.3±0.5 ng insulin/ml versus 4.5±0.5 ng/ml at t=5 min, P<0.001) and elevated blood glucose levels compared with those of the controls (29.19±1.3 mmol/l versus 23.5±1.7 mmol/l, P<0.05). In contrast, central i.c.v. injection of BIM23014C (0.75 µg) had no significant effect on either plasma insulin (3.3±0.4 ng/ml, P>0.05) or blood glucose levels (23.5±1.7 mmol/l, P>0.05) although i.v. administration of this dose increased blood glucose concentrations (32.3±0.7 mmol/l, P<0.01). BIM23014C did not measurably alter plasma glucagon, SST, GLP1 or catecholamine levels whether injected i.v. or i.c.v. These results indicate that SST does not suppress insulin secretion by a centrally mediated effect but acts peripherally on islet cells.

Reconceptualising the doctor-patient relationship: recognising the role of trust in contemporary health care.

The conception of the doctor-patient relationship under Australian law has followed British common law tradition whereby the relationship is founded in a contractual exchange. By contrast, this article presents a rationale and framework for an alternative model-a "Trust Model"-for implementation into law to more accurately reflect the contemporary therapeutic dynamic. The framework has four elements: (i) an assumption that professional conflicts (actual or perceived) with patient safety, motivated by financial or personal interests, should be avoided; (ii) an onus on doctors to disclose these conflicts; (iii) a proposed mechanism to contend with instances where doctors choose not to disclose; and (iv) sanctions for non-compliance with the regime.

Ablation of glucagon receptor signaling by peptide-based glucagon antagonists improves glucose tolerance in high fat fed mice.

Modification to the structure of glucagon has provided a number of glucagon receptor antagonists with possible therapeutic application for diabetes. These novel peptide analogs include desHis(1)Pro(4)Glu(9)-glucagon and desHis(1)Pro(4)Glu(9)(Lys(30)PAL)-glucagon. This study has evaluated the metabolic benefits of once daily administration of desHis(1)Pro(4)Glu(9)-glucagon and desHis(1)Pro(4)Glu(9)(Lys(30)PAL)-glucagon in high fat (45%) fed mice for 15 days. Administration of desHis(1)Pro(4)Glu(9)-glucagon and desHis(1)Pro(4)Glu(9)(Lys(30)PAL)-glucagon had no significant effect on body weight, food intake or circulating glucose concentrations during the treatment period. However, both peptides significantly (P<0.05 to P<0.01) reduced circulating plasma insulin concentrations from day 6 onwards. Oral glucose tolerance and insulin sensitivity, as assessed by exogenous insulin administration, were significantly (P<0.01 to P<0.001) improved by both desHis(1)Pro(4)Glu(9)-glucagon and desHis(1)Pro(4)Glu(9)(Lys(30)PAL)-glucagon. These metabolic benefits were accompanied by significantly (P<0.01) increased pancreatic insulin stores. No significant differences in blood triacylglycerol or cholesterol levels were noted with desHis(1)Pro(4)Glu(9)-glucagon, however desHis(1)Pro(4)Glu(9)(Lys(30)PAL)-glucagon treatment significantly (P<0.01) increased HDL-cholesterol levels. Glucagon-mediated elevations of glucose and insulin were effectively (P<0.01 to P<0.001) annulled in both treatment groups on day 15. Interestingly, glucose levels during an intraperitoneal glucose tolerance test were not altered by either desHis(1)Pro(4)Glu(9)-glucagon or desHis(1)Pro(4)Glu(9)(Lys(30)PAL)-glucagon treatment. These data provide further evidence that glucagon antagonism could provide an effective means of treating T2DM.

Metabolic phenotyping guidelines: assessing glucose homeostasis in rodent models.

The pathophysiology of diabetes as a disease is characterised by an inability to maintain normal glucose homeostasis. In type 1 diabetes, this is due to autoimmune destruction of the pancreatic ß-cells and subsequent lack of insulin production, and in type 2 diabetes it is due to a combination of both insulin resistance and an inability of the ß-cells to compensate adequately with increased insulin release. Animal models, in particular genetically modified mice, are increasingly being used to elucidate the mechanisms underlying both type 1 and type 2 diabetes, and as such the ability to study glucose homeostasis in vivo has become an essential tool. Several techniques exist for measuring different aspects of glucose tolerance and each of these methods has distinct advantages and disadvantages. Thus the appropriate methodology may vary from study to study depending on the desired end-points, the animal model, and other practical considerations. This review outlines the most commonly used techniques for assessing glucose tolerance in rodents and details the factors that should be taken into account in their use. Representative scenarios illustrating some of the practical considerations of designing in vivo experiments for the measurement of glucose homeostasis are also discussed.

Effects of short-term chemical ablation of glucagon signalling by peptide-based glucagon receptor antagonists on insulin secretion and glucose homeostasis in mice.

Glucagon is a hormone with important effects on blood glucose regulation. This study has utilized the stable glucagon receptor antagonists, desHis¹Pro4Glu9-glucagon and desHis¹Pro4Glu9(Lys¹²PAL)-glucagon, to evaluate the effects of sustained inhibition of glucagon receptor signalling in normal mice. Twice-daily injection of either analogue for 10 days had no effect on food intake, body weight and non-fasting plasma glucose concentrations. However, insulin levels were significantly raised (p<0.05 to p<0.01) from day 3 onwards in desHis¹Pro4Glu9-glucagon mice. After 10 days, glucose tolerance was improved (p<0.05) in desHis¹Pro4Glu9-glucagon treated mice. Glucose-mediated insulin secretion and circulating cholesterol levels were significantly (p<0.05 to p<0.01) decreased in both treatment groups. Importantly, the effects of glucagon to increase blood glucose and insulin concentrations were still annulled on day 10. Insulin sensitivity was almost identical in all groups of mice at the end of the study. In addition, no changes in pancreatic insulin and glucagon content or islet morphology were observed in either treatment group. Finally, acute injection of desHis¹Pro4Glu9-glucagon followed by a 24-h fast in treatment naïve mice was not associated with any hypoglycaemic episodes. These data indicate that peptide-based glucagon receptor antagonists represent safe and effective treatment options for type 2 diabetes.

desHis¹Glu9-glucagon-[mPEG] and desHis¹Glu9(Lys³°PAL)-glucagon: long-acting peptide-based PEGylated and acylated glucagon receptor antagonists with potential antidiabetic activity.

Glucagon is hormone secreted from the pancreatic alpha-cells that is involved in blood glucose regulation. As such, antagonism of glucagon receptor signalling represents an exciting approach for treating diabetes. To harness these beneficial metabolic effects, two novel glucagon analogues, desHis¹Glu9-glucagon-[mPEG] and desHis¹Glu9(Lys³°PAL)-glucagon, has been evaluated for potential glucagon receptor antagonistic properties. Both novel peptides were completely resistant to enzymatic breakdown and significantly (P<0.05 to P<0.001) inhibited glucagon-mediated elevations of cAMP production in glucagon receptor transfected cells. Similarly, desHis¹Glu9-glucagon-[mPEG] and desHis¹Glu9(Lys³°PAL)-glucagon effectively antagonised glucagon-induced increases of insulin secretion from BRIN BD11 cells. When administered acutely to normal, high fat fed or ob/ob mice, both analogues had no significant effects on overall blood glucose or plasma insulin levels when compared to saline treated controls. However, desHis¹Glu9-glucagon-[mPEG] significantly (P<0.05) annulled glucagon-induced increases in blood glucose and plasma insulin levels in normal mice and had similar non-significant tendencies in high fat and ob/ob mice. In addition, desHis¹Glu9(Lys³°PAL)-glucagon effectively (P<0.05 to P<0.001) antagonised glucagon-mediated elevations of blood glucose levels in high fat fed and ob/ob mice, but was less efficacious in normal mice. Further studies confirmed the significant persistent glucagon receptor antagonistic properties of both novel enzyme-resistant analogues 4h post administration in normal mice. These studies emphasise the potential of longer-acting peptide-based glucagon receptor antagonists, and particularly acylated versions, for the treatment of diabetes.

The role of NELL-1, a growth factor associated with craniosynostosis, in promoting bone regeneration.

Efforts to enhance bone regeneration in orthopedic and dental cases have grown steadily for the past decade, in line with increasingly sophisticated regenerative medicine. To meet the unprecedented demand for novel osteospecific growth factors with fewer adverse effects compared with those of existing adjuncts such as BMPs, our group has identified a craniosynostosis-associated secreted molecule, NELL-1, which is a potent growth factor that is highly specific to the osteochondral lineage, and has demonstrated robust induction of bone in multiple in vivo models from rodents to pre-clinical large animals. NELL-1 is preferentially expressed in osteoblasts under direct transcriptional control of Runx2, and is well-regulated during skeletal development. NELL-1/Nell-1 can promote orthotopic bone regeneration via either intramembranous or endochondral ossification, both within and outside of the craniofacial complex. Unlike BMP-2, Nell-1 cannot initiate ectopic bone formation in muscle, but can induce bone marrow stromal cells (BMSCs) to form bone in a mouse muscle pouch model, exhibiting specificity that BMPs lack. In addition, synergistic osteogenic effects of Nell-1 and BMP combotherapy have been observed, and are likely due to distinct differences in their signaling pathways. NELL-1's unique role as a novel osteoinductive growth factor makes it an attractive alternative with promise for future clinical applications. [Note: NELL-1 and NELL-1 indicate the human gene and protein, respectively; Nell-1 and Nell-1 indicate the mouse gene and protein, respectively.]

Developing the stage of change approach for the reduction of work-related musculoskeletal disorders.

Musculoskeletal disorders remain the most prevalent form of occupational ill health, prompting examination of why attempts to manage the problem have been less successful than perhaps hoped. With a view to improving the efficacy of such interventions, this study examined the applicability of the stage of change approach to the workplace. Tools were developed to assess individual and organizational stage of change towards reducing the risks of musculoskeletal disorders. The tools were tested in a range of occupational sectors, and demonstrated high levels of validity and reliability. Implications for the application of the model in the workplace for improving intervention effectiveness are discussed.

Electrostatic micromachine scanning mirror for optical coherence tomography.

Compact electrostatic micromirror structures for use in the scanning arm of an optical coherence tomography (OCT) system are described. These devices consist of millimeter-scale mirrors resting upon micrometer-scale polyimide hinges that are tilted by a linear micromachine actuator, the integrated force array (IFA). The IFA is a network of deformable capacitor cells that electrostatically contract with an applied voltage. The support structures, hinges, and actuators are fabricated by photolithography from polyimide-upon-silicon wafers. These devices were inserted into the scanning arm of an experimental OCT imaging system to produce in vitro and in vivo images at frame rates of 4 to 8 Hz.

Diverse spatial expression patterns of UDP-GalNAc:polypeptide N-acetylgalactosaminyl-transferase family member mRNAs during mouse development.

Cell migration and adhesion during embryonic development are complex processes which likely involve interactions among cell-surface carbohydrates. While considerable work has implicated proteoglycans in a wide range of developmental events, only limited attention has been directed towards understanding the 7role(s) played by the related class of mucin-type O-glycans. The initial step of mammalian mucin-type O-glycosylation is catalyzed by a family of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (ppGaNTases). The spatial expression patterns of the messenger RNAs of seven ppGaNTase family members were investigated from gastrulation through organogenesis stages of mouse development. The seven glycosyltransferases were expressed in unique patterns during embryogenesis. ppGaNTase-T1, -T2, -T4, and -T9 were expressed more ubiquitously than ppGaNTase-T3, -T5, and -T7. Organ systems with discrete accumulation patterns of ppGaNTase family members include the gastrointestinal tract (intestine, liver, stomach, submandibular gland), nervous system (brain, eye), lung, bone, yolk sac, and developing craniofacial region. The pattern in the craniofacial region included differential expression by family members in developing mandible, teeth, tongue and discrete regions of the brain including the pons and migratory, differentiating neurons. Additionally, ppGaNTase-T5 accumulates in a subset of mesenchymal cells at the ventral-most portions of the E12.5 maxilla and mandible underlying the dental lamina. The unique spatiotemporal expression of the different ppGaNTase family members during development suggests unique roles for each of these gene products.

Intracardiac ultrasound scanner using a micromachine (MEMS) actuator.

Catheter-based intracardiac ultrasound offers the potential for improved guidance of interventional cardiac procedures. The objective of this research is the development of catheter-based mechanical sector scanners incorporating high frequency ultrasound transducers operating at frequencies up to 20 MHz. The authors' current transducer assembly consists of a single 1.75 mm by 1.75 mm, 20 MHz, PZT element mounted on a 2 mm by 2 mm square, 75 mum thick polyimide table that pivots on 3-mum thick gold plated polyimide hinges. The hinges also serve as the electrical connections to the transducer. This table-mounted transducer is tilted using a miniature linear actuator to produce a sector scan. This linear actuator is an integrated force array (IFA), which is an example of a micromachine, i.e., a microelectromechanical system (MEMS). The IFA is a thin (2.2 mum) polyimide membrane, which consists of a network of hundreds of thousands of micron scale deformable capacitors made from pairs of metallized polyimide plates. IFAs contract with an applied voltage of 30-120 V and have been shown to produce strains as large as 20% and forces of up to 8 dynes. The prototype transducer and actuator assembly was fabricated and interfaced with a GagePCI analog to digital conversion board digitizing 12 bit samples at a rate of 100 MSamples/second housed in a personal computer to create a single channel ultrasound scanner. The deflection of the table transducer in a low viscosity insulating fluid (HFE 7100, 3M) is up to +/-10 degrees at scan rates of 10-60 Hz. Software has been developed to produce real-time sector scans on the PC monitor.

The role of carbohydrates in mammalian sperm-egg interactions: how important are carbohydrate epitopes?

Evidence implicating the involvement of carbohydrates in fertilization has been reported for decades in species which span the phylogenetic scale. The exact nature and role of these ligands in fertilization, however, has eluded investigators. Here, such investigations are reviewed as they relate to mammalian fertilization, with the principle focus on reviewing the role of carbohydrates involved in the primary binding event between sperm cell and egg.

A cytoplasmic protein, bystin, interacts with trophinin, tastin, and cytokeratin and may be involved in trophinin-mediated cell adhesion between trophoblast and endometrial epithelial cells.

Trophinin and tastin form a cell adhesion molecule complex that potentially mediates an initial attachment of the blastocyst to uterine epithelial cells at the time of implantation. Trophinin and tastin, however, do not directly bind to each other, suggesting the presence of an intermediary protein. The present study identifies a cytoplasmic protein, named bystin, that directly binds trophinin and tastin. Bystin consists of 306 amino acid residues and is predicted to contain tyrosine, serine, and threonine residues in contexts conforming to motifs for phosphorylation by protein kinases. Database searches revealed a 53% identity of the predicted peptide sequence with the Drosophila bys (mrr) gene. Direct protein-protein interactions of trophinin, tastin, and bystin analyzed by yeast two-hybrid assays and by in vitro protein binding assays indicated that binding between bystin and trophinin and between bystin and tastin is enhanced when cytokeratin 8 and 18 are present as the third molecule. Immunocytochemistry of bystin showed that bystin colocalizes with trophinin, tastin, and cytokeratins in a human trophoblastic teratocarcinoma cell, HT-H. It is therefore possible that these molecules form a complex and thus are involved in the process of embryo implantation.

Cloning and expression of mouse UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase-T3.

A novel isoform of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase, designated ppGaNTase-T3, has been cloned from a mouse testis cDNA library and expressed in COS7 cells. ppGaNTase-T3 displayed 64 and 59% amino acid identity with ppGaNTase-T1 and ppGaNTase-T2, respectively, and 96% amino acid identity with the recently reported human form of ppGaNTase-T3. The ppGaNTase-T3 transcript is abundant in the major salivary glands, gastrointestinal tract and both the male and female reproductive systems. ppGaNTase-T3 and ppGaNTase-T1 display overlapping substrate preferences in vitro, although mapping studies of O-glycosylated peptides suggests that certain hydroxyamino acids are preferentially glycosylated by each isoform. This suggests that more than one isoform of ppGaNTase may be required to complete the O-glycosylation of endogenous substrates.

Cobra venom factor immunoconjugates: effects of carbohydrate-directed versus amino group-directed conjugation.

Human IgM monoclonal antibody 16-88, derived from patients immunized with autologous colon carcinoma cells, was derivatized with two different cross-linkers, S-(2-thiopyridyl)-L-cysteine hydrazide (TPCH), which is carbohydrate-directed, and N-succinimidyl-3-(2- pyridyldithio)propionate (SPDP), which is amino group-directed. Two antibody functions, antigen binding and complement activation, were assayed upon derivatization with TPCH and SPDP. TPCH allowed for extensive modification (up to 17 TPCH molecules per antibody) without impairment of antigen binding activity, while this function was significantly compromised upon derivatization with SPDP. Antibody molecules derivatized with 16 SPDP residues showed almost complete loss of their antigen binding function. The complement activating ability of antibody 16-88 was significantly decreased after derivatization with TPCH or SPDP. In the case of SPDP derivatization, this decrease of the complement activating ability is predominantly a consequence of the impaired binding function. Upon conjugation of cobra venom factor (CVF), a nontoxic 137-kDa glycoprotein which is capable of activating the alternative pathway of complement, the antigen binding activity of SPDP-derivatized antibody was further compromised, whereas that of TPCH-derivatized antibody remained unaffected even after attachment of three or four CVF molecules per antibody. In both conjugates CVF retained good functional activity. CVF was slightly more active when attached to SPDP-derivatized antibody, suggesting a better accessibility of amino group-coupled CVF for its interaction with other complement proteins. These results indicate that carbohydrate-directed conjugation compromises the antibody function of complement activation, but allows for the generation of immunoconjugates with unimpaired antigen binding capability.(ABSTRACT TRUNCATED AT 250 WORDS)